Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effects on the migration of T-cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected Treg recruitment. These data suggest while age-related physical changes in the ECM can promote tumor cell motility, they may adversely impact the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older melanoma patients.

Older melanoma patients have lower rates of sentinel lymph node (LN) metastases yet paradoxically have inferior survival. Patient age correlated with an inability to retain Technetium radiotracer during sentinel LN biopsy in over 1000 patients, and high technecium counts correlated to better survival. We hypothesized that loss of integrity in the lymphatic vasculature due to ECM degradation might play a role. We have implicated HAPLN1 in age-dependent ECM degradation in the dermis. Here we queried whether HAPLN1 could be altered in the lymphatic ECM. Lymphatic HAPLN1 expression was prognostic of long-term patient survival. Adding rHAPLN1 to aged fibroblast ECMs in vitro reduced endothelial permeability via modulation of VE-Cadherin junctions, whereas endothelial permeability was increased following HAPLN1-knockdown in young fibroblasts. In vivo, reconstitution of HAPLN1 in aged mice increased the number of LN metastases, but reduced visceral metastases. These data suggest that age-related changes in ECM can contribute to impaired lymphatics.

Membranes made from electrospun nanofibers are potentially excellent for promoting bone growth for next-generation tissue scaffolds. The effectiveness of an electrospun membrane is shown here using high resolution 3D imaging to visualize the interaction between cells and the nanofibers within the membrane. Nanofibers that are aligned in one direction control cell growth at the surface of the membrane whereas random nanofibers cause cell growth into the membrane. Such observations are important and indicate that lateral cell growth at the membrane surface using aligned nanofibers could be used for rapid tissue repair whereas slower but more extensive tissue production is promoted by membranes containing random nanofibers.

The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of plectin facilitated greater nuclear deformation, which depended on actomyosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.

Highlights d Wnt5A increases the half-life of wild-type p53 to promote a slow-cycling phenotype d Multiple types of stress increase Wnt5A and p53 expression in metastatic melanoma d Inhibiting p53 sensitizes melanoma cells to BRAF/MEK

Dormant tumor cells escape the primary site, do not grow out into macroscopic tumors in the distal site, but maintain enough plasticity to reactivate and form overt metastatic lesions, sometimes taking several decades. Despite its importance in metastasis and residual disease, few studies have been able to successfully model or characterize dormancy within melanoma. Here, we show that age-related changes in the lung microenvironment facilitate a permissive niche for e cient outgrowth of disseminated dormant tumor cells, in contrast to the aged skin, where age-related changes suppress melanoma growth but drive dissemination. A model of melanoma progression that addresses these microenvironmental complexities is the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state 1-3 . Dermal broblasts are key orchestrators of promoting phenotype switching in melanoma via changes in the secretion of soluble factors during aging [4][5][6][7][8] . Speci cally, we have identi ed Wnt5A as a master regulator of activating metastatic dormancy, which enables e cient seeding and survival of melanoma cells in metastatic niches. Age-induced reprogramming of lung broblasts increases their secretion of the soluble Wnt antagonist sFRP1, which inhibits Wnt5A, enabling e cient metastatic outgrowth. Further, we have identi ed the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis respectively. Overall, we nd that age-induced changes in distal metastatic microenvironments promotes e cient reactivation of dormant melanoma cells in the lung. MainWe have previously established that melanoma cells implanted in aged mouse skin metastasize to the lung at greater rates than in younger animals 4 . Whether this is due to increased dissemination from the primary site, or because the aged microenvironment at metastatic sites promotes outgrowth remained unclear. To investigate this, we intradermally implanted Yumm1.7 (mCherry) melanoma cells into young (8 weeks) and aged (> 52 weeks) C57BL6 mice. The primary tumor in the skin grew faster in young mice (Fig. 1A). We examined distal lung metastases at weeks 1, 3 and 5 using immunohistochemical (IHC) analysis of mCherry positive cells in the lung. At week 1, we failed to detect melanoma cells. At week 3, we found that melanoma cells e ciently seed the lung in equal numbers in young and aged mice (Fig. 1B) as single cell colonies (Fig. 1C, top panels); however, at week 5, larger metastatic colonies formed in the aged lung (Fig. 1C, bottom right), while cells persisted as single cells in the young (Fig. 1C, bottom left). While the number of cells that seeded in the young vs. aged lung are similar, the rate at which cells seed the lung (no. of cells disseminating/mm 3 of tumor volume) was far lower in young mice. Thus, to determine whether the difference in lung outgrowth at week 5 was due to an overall increase in dissemination from aged primary tumors, we removed the primary tumors from aged m...

This review will focus on the role of the tumor microenvironment (TME) in the development of drug resistance in melanoma. Resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma is observed months after treatment, a phenomenon that is often attributed to the incredible plasticity of melanoma cells but may also depend on the TME. The TME is unique in its cellular composition-it contains fibroblasts, immune cells, endothelial cells, adipocytes, and among others. In addition, the TME provides "non-homeostatic" levels of oxygen, nutrients (hypoxia and metabolic stress), and extracellular matrix proteins, creating a pro-tumorigenic niche that drives resistance to MAPKi treatment. In this review, we will focus on how changes in the tumor microenvironment regulate MAPKi resistance.

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